Human mesenchymal stem cell therapy promotes retinal ganglion cell survival and target reconnection after optic nerve crush in adult rats

Abstract

Background: Optic-nerve injury results in impaired transmission of visual signals to central targets and leads to the death of retinal ganglion cells (RGCs) and irreversible vision loss. Therapies with mesenchymal stem cells (MSCs) from different sources have been used experimentally to increase survival and regeneration of RGCs.

Methods: We investigated the efficacy of human umbilical Wharton's jelly-derived MSCs (hWJ-MSCs) and their extracellular vesicles (EVs) in a rat model of optic nerve crush.

Results: hWJ-MSCs had a sustained neuroprotective effect on RGCs for 14, 60, and 120 days after optic nerve crush. The same effect was obtained using serum-deprived hWJ-MSCs, whereas transplantation of EVs obtained from those cells was ineffective. Treatment with hWJ-MSCs also promoted axonal regeneration along the optic nerve and reinnervation of visual targets 120 days after crush.

Conclusions: The observations showed that this treatment with human-derived MSCs promoted sustained neuroprotection and regeneration of RGCs after optic nerve injury. These findings highlight the possibility to use cell therapy to preserve neurons and to promote axon regeneration, using a reliable source of human MSCs.

Keywords: Cell therapy; Central nervous system; Mesenchymal stem cells; Nerve regeneration; Neuroprotection; Optic nerve injury.

Fig. 1

Survival of RGCs 14 days after crush and hWJ-MSC-based treatments. a–e Confocal images of an optical slice of whole-mounted retinas labeled for Tuj1, 14 days after crush and injection of vehicle, hWJ-MSCs, SD-hWJ-MSCs (after 24 h of fetal bovine serum deprivation), or EVs. Inset in the lower left corner of each image shows higher magnification of the dashed square. f Graph representing the number of RGCs of each experimental group normalized by control retinas. Mean ± SEM. One-way ANOVA with Tukey post-test, *P < 0.5, **P < 0.1, ***P < 0.001, ****P < 0.0001. Scale bars 50 μm for images and 21 μm for insets

Fig. 2

Long-term neuroprotective effect of hWJ-MSCs on RGCs. High magnification of confocal images of retinas immunostained for Tuj1 (a–d) 60 and 120 days after crush and injection of vehicle (a and b) or hWJ-MSCs (c and d). Arrows indicate RGCs with soma larger than 150 μm². e Number of RGCs in the ipsilateral retina normalized by the contralateral eye. f Percentage of surviving RGCs with soma larger than 150 μm² (black bars) at 60 and 120 days after crush and injection of vehicle or hWJ-MSCs. Mean ± SEM. Unpaired parametric t test, *P < 0.5. Scale bar 25 μm

Fig. 3

hWJ-MSC treatment promotes optic nerve regeneration. Photomontage of optic nerve sections immunostained for GAP-43 14 days after lesion and treatment with vehicle (a) or hWJ-MSCs (b). c Graph representing the mean ± SEM of axons at respective distances from the crush site in vehicle or hWJ-MSC-treated groups. Two-way ANOVA, **P < 0.01. Scale bar 200 μm

Fig. 4

hWJ-MSC treatment promotes long-term axonal regeneration. Photomontage of optic nerve sections labeled with CTB-Alexa555 120 days after lesion and treatment with vehicle (a) or hWJ-MSCs (b). b′, b″ Insets of b with arrow indicating individual axons. c Graph representing the mean ± SEM of axons at respective distances from crush site in the vehicle- or hWJ-MSC-treated groups. Two-way ANOVA, ***P < 0.001. Scale bars 200 μm for a and b; 40 μm for b′ and b″

Fig. 5

hWJ-MSC treatment promotes axonal regeneration up to the central nuclei. Representative photomontages of × 20 z-stack confocal images of coronal sections of the SC (a) and dLGN (b and b′) 120 days after optic nerve crush and injection of hWJ-MSCs. CTB labeling of the axonal terminals (red) and ToPro-3 nuclei (blue). Scale bars 200 μm (A); 25 μm (a–b′); 100 μm (b)

Fig. 6

RGC terminals activate SC neurons. Images of × 20 z-stack confocal images. NGFI-A immunolabeling to identify activation of SC cells after light exposure in coronal section of ipsi- and contralateral SC 120 days after optic nerve crush and treatment with vehicle (a, b) or hWJ-MSCs (c, d). Inset in d shows higher magnification of the dashed square. e Quantification of the number of cells expressing NGFI-A per mm² in each group ± SEM. Unpaired parametric t test, *P < 0.05, **P < 0.01. Scale bars 50 μm for images a–d; 23 μm for inset